The invention relates to macrolide chemistry. It concerns the compound of formula I 
i.e. {[1E-(1R,3R,4S)]1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R}-12-[2-(4-chloro-3-methoxycyclohexyl)-1-methylvinyl]-17-ethyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0(4,9)]octacos-18-ene-2,3,10,16-tetraone, hereinafter briefly named xe2x80x9c33-epichloro-33-desoxy-FR520xe2x80x9d or xe2x80x9c33-epichloro-33-desoxyascomycinxe2x80x9d, in crystalline form.
For simplicity, formula I as referred to herein should be understood as including the compound of formula I in the various tautomeric forms with which it is in equilibrum, particularly in solution, and solvated, e.g. hydrated forms, such as the tautomeric forms of formula 
and of formula 
The compound of formula I is known in amorphous form, e.g. from Sandoz EP 427 680, Example 66a in the form of a colourless foamy resin [with 1H-NMR=4.56 (m, H-33)], and from Merck EP 480 623, Example 53 (without any physicochemical characterization). Various names and carbon atom numberings are used in the literature.
Prior to the present invention, the compound of formula I had never been recovered in crystalline form.
It appears that the presence of a halogen atom, especially chlorine in the cyclohexyl moiety of the molecule, particularly in the 4 position thereof (also marked as position 33 in formulae I and Ic herein), has an unfavourable effect on the crystallization properties of this structural class of compounds. Thus in EP 427 680 none of the halogenated final products is obtained in crystalline form, they are colourless foams or foamy resins, and characterized by their NMR spectra.
Similarly, in EP 480 623, which covers exclusively macrolide end products halogenated in the cyclohexyl moiety, none of the specific compounds disclosed is characterized by data indicative of crystallinity, such as a melting point; most end products therein are not characterized by any physicochemical data at all, and those that are characterized, are characterized by their mass spectra, which are not indicative as regards physical state; and none of the 4-chloro end products disclosed is characterized at all.
Further analogous macrolides halogenated in the cyclohexyl moiety are also disclosed in e.g. Fisons WO 91/13889, specifically, as Examples 42a), 42b) and 49a): the compounds therein are also not obtained in crystalline form, but recovered as a foam or an oil.
Overall, the 23-membered tricyclomacrolides derived from FK 506 are obtainable in crystalline form only with difficulty, if at all, as appears also from e.g. Merck WO 97/8182, concerning a macrolide compound having a basic substituent capable of forming salts, which could be obtained in crystalline form, but as a tartrate salt. The compound of the present invention is devoid of such a basic substituent.
It is thus surprising that crystallization of the compound of formula I has now been successfully achieved.
The invention concerns the compound of formula I in crystalline form. The crystalline form may appear as solvated, e.g. hydrated, or anhydrous form, or be a tautomer.
While the first recovery of the compound of formula I in crystalline form occurred several years after the first synthesis of the compound, initially obtained only in amorphous form, it has turned out that subsequently to its first crystallization, the compound could be induced to crystallize from the amorphous form quite readily. The crystalline material has thus now become easily accessible, using a variety of experimental conditions extending beyond the initially used recrystallization conditions, which involved the addition of water to an ethanolic solution of the amorphous compound.
The invention also concerns a process for the preparation of the compound of formula I, or a tautomeric or solvated form thereof, in crystalline form which comprises appropriately converting amorphous compound of formula I from a solution thereof under crystallization-inducing conditions.
It also concerns the compound of formula I, or a tautomeric or solvated form thereof, in crystalline form whenever prepared by that process, and the compound of formula I in a non-crystalline, e.g. in dissolved state, or a tautomeric or solvated form thereof, whenever produced from a crystalline form.
The process of the invention is effected in conventional manner. The precise conditions under which crystals are formed may now be empirically determined and a number of methods are suitable in practice, including the initial addition of water to an ethanolic solution of the compound of formula I in amorphous form.
Crystallization-inducing conditions normally involve the use of an appropriate crystallization-inducing solvent, such as methanol, ethanol, isopropanol or water or mixtures thereof. Conveniently, the amorphous compound is dissolved in the solvent at a temperature of normally at least 10xc2x0 C. The solution may be produced by dissolving in a solvent any one or more of amorphous forms of the compound, and solvates thereof, such as hydrates, methanolates, ethanolates, isopropanolates and acetonitrilates. Crystals may then be formed by conversion from solution, crystallization taking place at a temperature of between about 10xc2x0 C. and the boiling point of the solvent. The dissolution and crystallization may be carried out in various conventional ways. For instance, amorphous compound may be dissolved in a solvent or a mixture of solvents in which it is readily soluble at elevated temperatures but in which it is only sparingly soluble at lower temperatures. Dissolution at elevated temperature is followed by cooling during which the desired crystals crystallize out of solution. Solvents which are suitable include esters such as methyl acetate and ethyl acetate, toluene and acetonitrile. Mixed solvents comprising a good solvent in which the compound is readily soluble, preferably, in amounts of at least 1% by weight at 30xc2x0 C., and a poor solvent in which it is more sparingly soluble, preferably in amounts of not more than about 0.01% by weight at 30xc2x0 C., may also be employed provided that crystallization from the mixture at a reduced temperature, of normally at least about, 10xc2x0 C., is possible using the selected solvent mixture.
Alternatively, the difference in solubility of the crystals in different solvents may be used. For example, the amorphous compound may be dissolved in a good solvent in which it is highly soluble such as one in which it is soluble in amounts of at least 1% by weight at about 30xc2x0 C., and the solution subsequently mixed with a poor solvent in which it is more sparingly soluble, such as one in which it is soluble in amounts of not more than about 0.01% by weight at about 30xc2x0 C. Thus, the solution of the compound in the good solvent may be added to the poor solvent, while maintaining normally a temperature in excess of about 10xc2x0 C., or the poor solvent may be added to the solution of the compound in the good solvent, again while normally maintaining a temperature in excess of about 10xc2x0 C. Examples of good solvents include lower alcohols, such as methanol, ethanol and isopropanol, as well as acetone, tetrahydrofuran and dioxane. Examples of poor solvents are water, hexane and diethyl ether. Preferably, crystallization is effected at a temperature in the range of about 10xc2x0 C. to about 60xc2x0 C.
In an alternative embodiment of the process of the invention, solid amorphous compound is suspended at a temperature of normally at least about 10xc2x0 C. in a solvent in which it is incompletely soluble, preferably only sparingly soluble, at that temperature. A suspension results in which particles of solid are dispersed, and remain incompletely dissolved in the solvent. Preferably the solids are maintained in a state of suspension by agitation e.g. by shaking or stirring. The suspension is kept at a temperature of normally about 10xc2x0 C. or higher in order to effect a transformation of the starting solids into crystals. The amorphous solid compound suspended in a suitable solvent may be a solvate, e.g. hydrate, methanolate, ethanolate, isopropanolate or acetonitrilate. The amorphous powder may be derived by drying a solvate.
It is preferred to add xe2x80x9cseedsxe2x80x9d of crystalline material to the solution in order to induce crystallization.
The compound of formula I in crystalline form can readily be isolated, it can e.g. be filtered off or centrifuged from the crystallization medium, if desired after cooling, and washed and dried, and optionally further recrystallized using similar conditions.
While the initial recovery has resulted in material in a crystalline form designated as xe2x80x9cForm Axe2x80x9d herein, surprisingly, it has turned out upon further investigation that at least one additional crystal form of the compound may be recovered, herein designated as xe2x80x9cForm Bxe2x80x9d, which differs from Form A in various characteristics, such as its solubility. The invention thus concerns the compound of formula I or a tautomeric or solvated form thereof in crystalline form as such, and more particularly Form A and Form B. Form A is preferred.
Form A normally is in hydrated form at room temperature. The hydrated form can be reversibly dehydrated by heating to about 110xc2x0 C. It remains in Form A thereby. The hydrated form is the more stable state of Form A at room temperature. Form B normally is not in hydrated form, even at room temperature. It is thermodynamically a more stable form than Form A.
A crystal form is defined herein as being xe2x80x9ccrystallographically purexe2x80x9d when it contains at most about 0.5% (w/w), e.g. at most about 0.1% (w/w) of other form. Thus e.g. xe2x80x9ccrystallographically pure Form Axe2x80x9d contains about xe2x89xa60.5% (w/w), .g. about xe2x89xa60.1% (w/w) of Form B and/or amorphous form.
The preparation of Forms A and B may be effected using conventional means, starting either from amorphous material or from Form B or Form A, respectively, or mixtures thereof. Normally, the starting material is dissolved into an appropriate solvent and crystallized or recrystallized therefrom under conditions preferentially producing either Form A or Form B, resulting in crystallographically pure Form A or Form B.
The invention thus includes a process variant for the preparation of the compound of formula I, or a tautomeric or solvated form thereof, in crystalline Form A which comprises appropriately converting compound of formula I in other than Form A, or a tautomeric or solvated form thereof, from a solution thereof under conditions inducing preferential crystallization of Form A. It also concerns the compound of formula I in Form A whenever prepared by that process variant.
Conversely, the invention includes a process variant for the preparation of the compound of formula I, or a tautomeric or solvated form thereof, in crystalline Form B which comprises appropriately converting compound of formula I in other than Form B from a solution thereof under conditions inducing preferential crystallization of Form B. It also concerns the compound of formula I in Form B whenever prepared by that process variant.
For the preparation of Form A the starting material is conveniently dissolved in an appropriate solvent, preferably ethanol/water, preferably in the proportions 9.5:0.5. The temperature for dissolution is from about 60xc2x0 C. to about 75xc2x0 C., preferably about 70xc2x0 C. The proportion of starting material to solvent preferably is from about 1:5 to about 1:6 on a weight basis, preferably about 1:5 (w/w). The solution is filtered and then cooled to a reduced temperature, preferably of from about 70xc2x0 C. to about 20xc2x0 C., preferably about 10xc2x0 C., and a liquid in which Form A is insoluble, such as water, is carefully added. A supersaturated solution results thereby. While crystals of Form A may spontaneously be formed, preferably the supersaturated solution is seeded with a few crystals of crystallographically pure Form A. It is usually beneficial to check the purity of the seeding crystals with a melt microscope. Further addition of liquid under careful stirring leads to more crystals of Form A. Low temperature, i.e. below about 20xc2x0 C., and seeding with crystallographically pure crystals of Form A appear to prevent the formation of crystals of Form B. Too lengthy stirring may be counter-productive, particularly at temperatures above 10xc2x0 C., Form B being the thermodynamically more stable form.
Conveniently, as a preliminary step, the starting material is preferably thoroughly dissolved in a polar organic solvent such as an alcohol, e.g. methanol, ethanol, isopropanol, preferably ethanol, or in acetone, especially in acetone, preferably at boiling temperature, and the solvent evaporated to dryness.
For the preparation of Form B the starting material is again dissolved in a solvent as described above for preparing Form A, preferably ethanol/water 9.5:0.5 (v/v). The temperature for dissolution is again from about 60xc2x0 C. to about 75xc2x0 C., preferably about 70xc2x0 C., and the resultant solution is filtered. The proportion of starting material to solvent is somewhat less than for preparing Form A, it is preferably about 1:7 (w/w). However, cooling is to a higher temperature than when preparing Form B, it is preferably to above 20xc2x0 C., e.g. to about 25xc2x0 or 30xc2x0 C., and the further workup is also effected at about that temperature or a similar temperature. Seeding with crystals of Form B is optional, but greatly facilitates crystallization and allows more latitude as regards e.g. temperature. The speed of formation of the supersaturated solution appears also to exert some effect on the result obtained, speedy supersaturation resulting in increased formation of Form B.
A solvate, e.g. a hydrate, may be converted into the corresponding unsolvated form in conventional manner and vice-versa, e.g. by appropriately heating up the solvated form, or cooling down the unsolvated form of the crystal form susceptible of being solvated.
The two crystal forms identified are characterized i.a. by the following physico-chemical data:
1) Form A:
appearance: white to off-white, finely crystalline powder (from ethanol/water);
m.p. determined by DSS (10xc2x0 K/mir): melting onset at about 132xc2x0 C.;
solubility (at 5xc2x0 C.):
water: insoluble
methanol, ethanol, ethyl acetate, diethylether, diisopropyl ether:  greater than 100 mg/ml
hexane:  less than 10 mg/ml;
solubility (at 25xc2x0 C.):
acetone, acetonitrile, ethanol, ethyl acetate, isopropanol, methanol:  greater than 50 mg/ml;
water:  less than 1 mg/ml;
solubility in the oil phase of cream (oleyl alcohol/miglyol 812R 4:6): 2.49%;
chemical purity: 98.5%;
thermogravimetry: loss of mass on drying up to melting: 1.46% (Carl Fischer titration);
morphology (SEM): sticks and agglomerates (1-100 xcexcm);
hygroscopicity (uptake determined by thermogravimetry): 1.49% (1 day, 92% r.h.); 1.78% (1 week, 25xc2x0 C., 75% r.h.);
DSC curve: see FIG. 1 (Perkin Elmer DSC-7 differential scanning calorimeter; measurement from 40xc2x0 C. to 200xc2x0 C., scan heating rate 10xc2x0 K/min);
FT-IR spectrum: see FIGS. 3 and 5 (PE FT-IR spectrometer 1725X; KBr, paraffin oil; scan range 4000-400 cmxe2x88x921);
X-ray powder diffraction pattern: see FIG. 6 (Scintag XDS 2000 powder diffractometer; Scintag, Santa Clara, Calif., USA); scan speed 0.5xc2x0 or 1xc2x0/min (2 theta value);
2) Form B:
m.p. determined by DSC (10xc2x0 K/min): melting onset at about 159xc2x0 C.;
solubility (at 5xc2x0 C.):
water: 0.3 mg/ml
methanol: 46.3 mg/ml
ethanol: 18.1 mg/ml
ethyl acetate:  greater than 50 mg/ml
diethylether: 9.3 mg/ml;
diisopropylether: 1.9 mg/ml
hexane: 0.8 mg/ml;
solubility (at 25xc2x0 C.):
water: 0.4 mg/ml
methanol:  greater than 50 mg/ml
ethanol: 34.4 mg/ml
ethyl acetate:  greater than 50 mg/ml
diethyl ether: 16.3 mg/ml
diisopropyl ether: 3.1 mg/ml
hexane: 1.5 mg/ml;
solubility in the oil phase of TMF cream (oleyl alcohol/myglyol 812R 4:6): 0.37%;
chemical purity: 99.9%;
thermogravimetry: loss of mass on drying up to melting:  less than 0.05%;
morphology (SEM): needles;
hygroscopicity(uptake determined by thermogravimetry): 1 day, 92% r.h. and 1 week, 25xc2x0 C., 75% r.h.: none;
DSC curve: see FIG. 2 (Perkin-Elmer DSC-7; 40xc2x0 C. to 200xc2x0 C.; scan heating rate 10xc2x0 K/min);
FT-IR spectrum: see FIGS. 4 and 5 (PE FT-IR spectrometer 1725X; KBr; paraffin oil; scan range 4000-400 cmxe2x88x921);
X-ray powder diffraction pattern: see FIG. 6 (Scintag XDS 2000 powder diffractometer); scan speed 0.5xc2x0 or 1xc2x0/min (2 theta value).
3) For reference, the corresponding FT-IR spectrum of the amorphous form is indicated in FIG. 7.
Characterization data for all forms of the compound is further as follows:
optical rotation: [xcex1]D20=xe2x88x9248.0xc2x0 (xc2x10.2xc2x0) (CDCl3);
TLC:
Rf=0.18 (silicagel; hexane/ethyl acetate 2:1)
Rf=0.62 (silicagel; hexane/ethyl acetate 1:1);
1H-NMR (CDCl3): Two conformers (Z:E=1:2). Characteristic signals of the major conformer d [ppm]: 5.35 (d,J=1.7 Hz, H-26, 5.12 (d,J=9.0 Hz, H-29), 5.05 (d,J=9.4 Hz, H-20), 4.60 (d,J=5.0 Hz, H-2), 4.56 (m,wxc2xd=10 Hz, H-33), 4.43 (d, J=13.8 Hz, H-6e), 3.66 (dd, J=9.6 Hz, J=1.0 Hz, H-14), 3.92 (m,H-24), 2.80 (dd, J=15.9 Hz, J=2.7 Hz, H-23a);
13C-NMR (CDCl3): Two conformers (Z:E=1:2). Signals of the major conformer d [ppm]: 213.7 (C-22), 196.3 (C-9), 169.1 (C-1), 164.8 (C-8), 138.8 (C-l9), 132.5 (C-28), 129.2 (C-29), 122.0 (C-20), 97.0 (C-10), 79.2 (C-32), 76.7 (C-26), 75.2 (C-15), 73.7 (C-14), 72.9 (C-13), 70.2 (C-24), 59.3 (C-33), 56.7 (C-2), 54.7 (C-21), 48.6 (C-18), 39.2 (C-6), 42.7 (C-23), 39.4 (C-25), 34.7 (C-30), 34.6 (C-11), 32.8 (C-12), 32.1 (C-35), 31.7 (C-34), 27.7 (C-3), 26.4 (C-17), 25.5 (C-31), 24.5 (C-5), 24.2 (C-36), 21.1 (C-4), 20.6 (17-Me), 16.2 (11-Me), 15.9 (19-Me), 14.2 (28-Me), 11.7 (C-37), 9.3 (25-Me).
In the above NMR spectra the carbon atom numbering is as appears in formula Ic hereafter: 
Abbreviations
DMSO: dimethylsulfoxide
DSC: differential scanning calorimetry
FT-IR: Fourier transformed infrared
m.p.: melting point
r.h.: relative humidity
SEM: scanning electron microscopy
T: transmission
TG: thermogravimetry
THF: tetrahydrofuran
TLC: thin layer chromatography